Because of the pathogenicity of Shiga I and Shiga II toxin producing bacteria (Sandvig, 2001; Cleary, 2004) and multiple recent food borne outbreaks (McCarthy, 1996; Koohmaraie et al., 2007; Cody et al., 1999; CDC, 2006; Denny et al., 2008; Snedecker et al., 2008; Anonymous, 2009; Snedecker et al., 2009) in the United States and abroad involving E. coli O157 serovars that produce these toxins, the development of an analytical method for their rapid, specific detection in food is needed. Such a method should have a low cost per sample and be as sensitive as possible because as few as ten E. coli O157 cells can cause infection (FDA, 2009a and 2011).
Flow cytometry has been used to measure cellular RNA and DNA content (Berdalet and Dortch, 1991), diagnose health disorders (Muirhead, 1984), determine cell viability (Comas and Vives-Rego, 1998) and quantify protein expression (Hedhammer et al., 2005). The use of flow cytometry to identify food borne pathogenic bacteria, such as Escherichia coli serotype O157, has been proposed by several groups (Tortorello et al., 1998; Kusunoki et al., 2000; Leach et al., 2010; Yang et al., 2010). Because of flow cytometry's sensitivity for characterizing individual particles, and its ability to detect target cells (for example to determine their identity and determine if they are viable) without culturing the cells can provide an advantage to other methods, such as PCR which can amplify DNA from non-viable cells (thus making it difficult to distinguish live from dead cells) and also can be inhibited in some food matrices (Rossen, 1992).
For environmental samples that do not have a large amount of background contamination or for applications where an ultralow detection limit is not necessary, a sample can be analyzed in minutes. Flow cytometry measurements can distinguish target bacteria in small numbers from non-target cells and other debris. However, when the application requires finding a few very pathogenic cells, there is the challenge of growing them into a number sufficient to detect and count the population of interest above matrix background. This is the situation for pathogenic E. coli O157, especially in food. Analysis of such requires an arsenal of appropriate sample preparation techniques.
The analysis of spinach (Spinacia oleracea) is difficult because of background interference (Leach et al., 2010), for example from endogenous chlorophylls, carotenes, iron heme-containing proteins, flavinoids and other biomolecules that absorb and emit light (Green and Durnford, 1996; Gil et al., 1999) in the critical FL1 (green) channel used for enumerating cells. Methods are needed to increase the counts-to-threshold ratio, C/T to enable near-real-time analysis in food with reliable detection of low levels of bacterial contamination. In addition, methods that can be completed within 8 hours, a normal packaging or production plant shift, are needed. Such speed and sensitivity can allow near-real-time QA/QC and not just retrospective determination, results currently available only after multiple plant decontamination cycles.
Most current bacterial diagnostic methods utilize sample volumes between 100 and 500 μL. Small sample volumes, coupled with instrumental limits of detection (LOD) of far more than 1 CFU, require concentration and/or enrichment of samples to facilitate meaningful analysis. As of April, 2010, 21 commercially available rapid analytical methods were performance tested for detection of E. coli O157 in one or more foods, including ground beef, apple cider, orange juice, pasteurized milk, spinach, lettuce, and boneless beef trim (AOAC International (2010) AOAC Performance Tested methods. www.aoac.org/testkits/testedmethods.html). Most of these methods specify a selective liquid culture first step designed to depress growth of background microflora while permitting that of the target pathogen (Amaguana et al., (1998) J. AOACI 81:721-6; Hammack et al., (2003) J. AOACI 86:714-8; DuPont (2010) DuPont Qualicon BAX System Enrichment media for E. coli O157:H7MP, www2.dupont.com/Qualicon/en_US/assets/downloads/BAX %20product %20descrip-Ecolistd.pdf; bioMérieux, Inc. (2009) VIDASC) ECO & VIDAS ICE, Protocol validated by AFNOR AOAC RI Performance TestedSM Method Certificate No. 010502, VID-006-09 www.biomerieux-usa.com). The most recent US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) method for E. coli O157 specifies a 3 hour first step non-selective enrichment to resuscitate injured target cells followed by enrichment for 20 hours in double-strength tryptone phosphate broth (Feng & Weagant, (2009) US FDA Bacteriological Analytical Manual. Chapter 4a, Diarrheagenic Escherichia coli). Growth in liquid media also serves to dilute endogenous food constituents or additives that may inhibit the analysis or otherwise interfere with the assay and to demonstrate by replication that detected target cells are viable. On the other hand, enrichment not only lengthens time-to-results (TTR) but also may not be effective using selective media. In the last fifteen years it has been recognized that bacteria under stress may be “viable but not culturable”, retaining the ability to cause disease, even when attempted culture from contaminated food has failed (Oliver, (2005) J. Microbiol. 43 (Spec. No.), 93-100).
The FDA evaluated the growth of E. coli O157 strains inoculated at low levels (0.12 to 0.42 CFU/g) into alfalfa sprouts and subsequently grown in a variety of selective media typically used at the first stage of analysis by conventional BAM or rapid methods (Weagant & Bound, Int. J. Food Microbiol. 71: 87-92, 2001). The results showed that using any of these selective media for recovery of E. coli O157 from alfalfa and mixed salad sprouts required a minimum growth period of 24 hours. Attempts to recover target cells in 6 hours failed in many cases. The reasons for the failure included the presence of bacterial growth inhibitors in the sprouts and antibiotics or other bactericides in the selective culture media. In tests not associated with food additives or other environmental stresses, the antibiotics did not depress growth for most strains. When a small number of stressed but viable pathogenic cells were present, there was a significant probability that none would recover and multiply during the shorter period (Kaprelyants and Kell, (1996) Trend. Microbiol., 4:237-242).